The natural killer activity of peritoneal fluid lymphocytes is decreased in women with endometriosis*

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1 FERTILITY AND STERILITY Copyright <> 1992 The American Fertility Society Printed on ocid-free paper in U.S.A. The natural killer activity of peritoneal fluid lymphocytes is decreased in women with endometriosis* Didier J. Oosterlynck, M.D.t+ Christel Meuleman, M.D.:!: Mark Waer, M.D., Ph.D.t Michel Vandeputte, M.D., Ph.D.t Philippe R. Koninckx, M.D., Ph.D.:!: Rega Institute for Medical Research, University of Leuven, Leuven, Belgium Objective: To investigate the local natural killer (NK) activity of the peritoneal fluid mononuclear cells (PFMC). Design, Patients: In a prospective way, the NK activity (K562-assay) was measured in peripheral blood (PB) and peritoneal fluid (PF) of 44 women who underwent a laparoscopy for infertility and/ or pain at the University Hospital of Gasthuisberg, Leuven, Belgium. Main Outcome Measure: The NK activity of peripheral blood mononuclear cells and PFMC, the number and concentration of PFMC, the percentage of lymphocytes versus macrophages by May-Griinwald-Giemsa staining and the estradiol and progesterone concentration of the PF were correlated together and with the severity of endometriosis. Results: We demonstrated that there is a significant NK activity in PF and that this activity is decreased in women with endometriosis. This defect was more pronounced in the follicular phase of the cycle compared with the postovulatory phase. In PB of the same 44 women, the decreased NK activity correlated with the severity of the disease. This confirms our previous report on another 34 women. Conclusions: The NK activity is decreased in women with endometriosis and correlated significantly with the severity of the disease in both the PB and PF of women with endometriosis. Fertil Steril 1992;58:290-5 Key Words: Endometriosis, peritoneal fluid, natural killer activity Peritoneal fluid (PF) has been reported to contain approximately 1 X 10 6 cells/ml, 85% of which are macrophages and 15% desquamated mesothelial cells and lymphocytes (1). Several studies have focused on the macrophages, being the most numerous subpopulation in the PF. In the PF of women with endometriosis, an increased number, concentration, Received January 2, 1992; revised and accepted April 8, * Supported by the National Fund for Scientific Research, Brussels, Belgium. t Rega Institute for Medical Research. * Department of Obstetrics and Gynecology, University of Leuven. Reprint requests: Didier Oosterlynck, M.D., Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium. and activation of these macrophages has been described by most authors (2-7), although some reported conflicting results (8). The macrophages in the peritoneal cavity are considered to result from a local irritation induced by the endometrial implants or by retrograde menstruation through the tubes. They were mainly investigated as a possible mechanism to explain the infertility in women with endometriosis. Indeed, macrophages have been described to influence ovulation, to inhibit ovum pickup, and to impair early embryo survival (9). Furthermore, it has been demonstrated that macrophages phagocyte spermatozoa, whereas toxic PF products decrease sperm motility (9). In a previous study, we showed a decreased lymphocyte mediated cytotoxicity toward autologous endometrial cells in women with endometriosis (10). 290 Oosterlynck et al. NK activity in PF Fertility and Sterility

2 This was because of a defective natural killer (NK) activity in the peripheral blood (PB) of those women. Natural killer cells have a spontaneous cytotoxicity against a wide variety of both autologous and allogeneic target cells. Natural killer cells can lyse these target cells without previous sensitization and without the involvement of major histocompatibility antigens. Descriptive studies already showed the presence of NK cells in PF (11). We now wanted to investigate the functional activity of those peritoneal NK cells in women with and without endometriosis. This local NK activity in the PF may be important in preventing implantation of free endometrial cells after menses. MATERIALS AND METHODS Patients and Fluid Collection Peritoneal fluids were obtained from 39 consecutive women undergoing a diagnostic laparoscopy for infertility (n = 28), for pain (n = 6), or for both (n = 5) at the Division of Reproductive Medicine, Department of Obstetrics and Gynecology, University Hospital Gasthuisberg, Leuven. Five women undergoing a laparoscopical tubal sterilization, after achieved parenthood, were also included to increase the number of women without endometriosis in this study. N one of the women received any hormonal treatment during the 3 months preceding the intervention. The day of the cycle was determined by the 1st day of their last menstrual period (LMP) during the follicular phase and by the basal body temperature (BBT) or by endometrial biopsy dating during the luteal phase. Endometriosis was staged according to the revised American Fertility Society classification (12). Endometriosis was found in 23 women and scored as: AFS I (n = 9), AFS II (n = 8), and AFS III and IV (n = 6). In 21 women no endometriosis was found. In 17 women, the endometriotic lesions were excised with CO 2 laser endoscopy, as described previously (13) and histologically confirmed. In one woman, however, with a deep lesion in the pouch of Douglas, together with a presumed endometrioma, the latter turned out to be a hemorrhagic corpus luteum at histology. In the other 6 women with endometriosis, the implants were vaporized with the CO 2 laser. Immediately before laparoscopy 10 ml of peri pheral venous blood was collected. At the beginning of the procedure, after insertion of the trocar, all the PF was aspirated through the laparoscope from the posterior cul-de-sac. These fluids were collected in sterile heparinized tubes at room temperature. The volume and the color were measured, and bloody fluids were excluded from the study (n = 5). Fluids were transferred immediately to the laboratory and processed within 30 minutes. The fluids were centrifuged, (400 X g for 10 minutes) and the supernatants stored at -20 C until assayed. The pellet was resuspended in minimal essential medium (MEM, GIBCO, Grand Island, NY), and white blood cells were counted manually. The viability was checked with trypan blue exclusion test. The heparinized PB, as well as the PF cells suspended in MEM, were centrifuged on Ficoll (Lymphoprep TM; Nyegaard, Oslo, Norway) to harvest a suspension free of granulocytes and red blood cells (RBCs) for the functional assays. In vitro depletion of macrophages was obtained by incubating the PF mononuclear cells (MC) during 1 hour at 37 C in plastic Petri dishes. The nonadherent PFMC were harvested after this incubation and resuspended in Roswell Park Memorial Institute (RPMI) medium (GIBCO) + 20% fetal calf serum (FCS, GIBCO) at the concentration of 5 X 10 6 cells/ml and used in the functional assay. Differential Count of WBCs The percentage of lymphocytes and macrophages in the final suspension used for the functional assays was counted after May-Griinwald-Giemsa staining. Smears of the PF cells were made on a glass slide, air dried before staining, and >500 cells were counted at a magnification of 400X. Morphologically, the following MC types were identified: monocytes and large tissue macrophages were recognized by their large size, their multilobulated nucleus, and slightly vacuolated cytoplasm. The lymphocytes, on the other hand, with their high nuclear/cytoplasmic ratio presented as darker cells. Measurement of NK activity of the PFMC was done in the same manner as for the PBMC. Details of this cytotoxicity assay were described previously (10). Briefly, after Ficoll centrifugation peritoneal cells were washed two times and resuspended at the final concentration of 5 X 10 6 cells/ml in RPMI supplemented with 20% FCS. For the chromium release assay, 5 X 105 effector cells were assayed together with 1 X 10451Cr-labeled target (K562) cells, both in 200 ill RPMI with 20% FCS, for 4 hours at 37 C in 5% CO 2, After this period, the released 51Cr radioactivity was measured in 100 ill supernatants of each well after centrifugation the microtiter plate at 400 X g for 2 minutes. The percentage of specific lysis was determinated as described before (10). To quantify the total NK Oosterlynck et al. NK activity in PF 291

3 activity in the PF, we multiplied for each woman the number of PFMC found after Ficoll centrifugation with the NK activity, as measured in each woman with an equal number of PFMC (5 X 10 5 PFMC/cup). The NK activity per ml of PF was measured by multiplying the specific immune release (SIR) of the K562-assay using PFMC with the number of PFMC per ml after Ficoll centrifugation (Table 1). Hormone Concentrations in PF Estradiol (E 2 ), progesterone (P), and CA-125 in the PF were measured with commercial radioimmunoassay kits. Estradiol was measured using the kit of Biomerieux (Brussels, Belgium). Progesterone was analyzed with the kit of Medgenix (Craponne, France). For CA-125 the kit of Centocor (Malvern, P A) was used and assayed using serial dilutions in male plasma (14). Statistical Analysis Statistical evaluation of the data was done with statistical analysis system. Student's t-test, ANOVA (Kruskal-Wallis for nonparametric evaluation), Pearson's, and Spearman's correlation were used where appropriate. RESULTS Natural Killer Activity (K562-Assay) The NK activity of PBMC (Table 1) is decreased in women with endometriosis and correlates with the severity of the disease (ANOV A, P = 0.002). The results of the present study, including 44 new women, are similar to those of our previous study on 34 women, confirming the presence of a deficient NK activity in the PB of women with endometriosis. With PFMC as effector cells against K562 target cells, the deficient NK activity in women with endometriosis was even more significant (ANOV A, P = ). Indeed, the NK activity mediated by PFMC of women with endometriosis (n = 23) was decreased to 31.1 % of the cytotoxicity mediated by PFMC of women without endometriosis (n = 21). Using PBMC, on the other hand, the cytotoxicity decreased only to 74.3% ofthe control cytotoxicity. The 21 women without endometriosis of the control group can be subdivided into two groups, depending on the presence of infertility. Thirteen women did have infertility and 8 did not and received a laparoscopy for a tubal ligation (n = 5) or for a pain problem (n = 3). The NK activity of the PBMC was 59.2% ± 9.1% compared with 52.7% ± 11.6% for the women with and without infertility, respectively. Using PFMC the NK activity was 15.1% ± 7.6% and 16.3% ± 7.2%, respectively. (These differences are not significant.) The number and concentration of PFMC in women with endometriosis (Table 1) were found to be increased, which is a well-known finding (2-7). To correct for this difference in number and concentration of PFMC, both the total NK activity in the PF and the NK activity per ml of PF were calculated. The results show that despite the increased number and concentration of PFM C in women with endometriosis the total NK activity and NK activity per ml of PF are still decreased in women with endometriosis. This correlated significantly with the severity of the disease (ANOV A, P = and 0.005, respectively). Table 1 Natural Killer Activity in PF and PB of 44 Women RevisedAFS No endometriosis I (n = 21) (n = 9) PBMC K562-assay* 56.2 ± ± 4.8 PFMC K562-assayt 15.5 ± ± 5.3 Numbert 5.4 ± ± 5.3 Concentration 0.54 ± ± 0.5 Total NKII 93.6 ± ± 46.2 NK/mL1l 9.1 ± ± 4.7 * K562-assay with PBMC; values are percent means ± SD of specific immune release. t K562-assay with PFMC; values are percent means ± SD of specific immune release. t Number of mononuclear cells after Ficoll centrifugation of PF; values are X10 6 cells. II III and IV (n = 8) (n = 6) ANOV A Probability 37.3 ± ± ± ± ± ± ± ± ± ± ± ± Concentration of MC after Ficoll centrifugation of PF; values are X10 6 celis/ml. II K562-assay of PFMC X number of cells after Ficoll centrifugation; the ANOVA is done on the logarithm of the value. 11 K562-assay of PFMC X concentration of cells after Ficoll centrifugation; the ANOVA is done on the logarithm of the value. 292 Oosterlynck et al. NK activity in PF Fertility and Sterility

4 Moreover, to exclude the possibility that an increase of the percentage of macrophages versus lymphocytes in the PF could be responsible for the decrease in NK activity, smears ofpfmc after Ficoll centrifugation were done in 27 women of this study. The percentage macrophages was 67% ± 12.1% in women without endometriosis (n = 11) versus 72.8% (±17% in women with endometriosis (n = 17). Student's t-test is not significant (P = 0.3). Natural Killer Activity of PFMC Is Not Influenced by Macrophages Depletion of macrophages was performed to verify whether these cells exert an inhibitory effect on the cytotoxic function of the lymphocytes against K562- cells. In three women without endometriosis, the K562-assay was performed with PFMC before and after in vitro depletion of the macrophages. The SIR before depletion was 34%, 26.6%, and 11.7% versus 28.2%, 30.5%, and 6.7% after in vitro depletion of macrophages in these three women, respectively. No obvious changes in NK activity were found, despite the marked decrease in macrophages after depletion. Indeed, the differential counts on May-Grunwald Giemsa stained smears showed that macrophages dropped from 81%, 74%, and 75% before to 35%, 37%, and 31 % after depletion, respectively. Natural Killer Activity During the Menstrual Cycle The E2 and P concentrations in PF increased sharply after ovulation and declined thereafter as described previously (15). CA-125 concentrations in PF were increased in the peri menstrual period, but there was no significant correlation with the functional assays (Table 2). No cyclic changes in the NK activity of the PBMC could be detected. With the PFMC the NK activity increased slightly in the early postovulatory phase of the cycle. These differences were significant when only the women with endometriosis were included. The NK activity of the PFMC measured on the 10 women during the postovulatory phase was 9.2 ± 3.5 compared with 4.6 ± 3 for the 13 women being in the follicular or late luteal phase of the menstrual cycle (Student's t-test, P = 0.005). The postovulatory increase in NK activity of the PFMC is not significant in those women. The E2 and P concentration correlated not with the absence or presence and severity of endometriosis but correlated positively with the NK activity of the PFMC, although significant values were not reached (Spearman, P = 0.09 and 0.16, respectively). Endometriosis and the Color of the PF The fluids used in this s-tudy were either strawcolored (n = 22), pink (n = 16), or red (n = 6). In women with endometriosis the PFs were more frequently blood stained. A significant correlation was found between the severity of endometriosis and the color of the PF (ANOV A, P = 0.01). Furthermore, we found an increased amount of bloody fluids in the proliferative phase of the cycle. The correlation between the day of the cycle and the color of the PF was significant (ANOV A, P = 0.01) but not between the day of the cycle and the severity of endometriosis (ANOVA, P = 0.34). DISCUSSION The pathophysiology of endometriosis has often been linked to immunological mechanisms over the last decade (16). According to this theory, viable endometrial cells that reach the peritoneal cavity by retrograde menstruation could implant more easily in women with a defective immunological defense Table 2 Hormone Concentrations in PF and K562-Assay Cycle day* 1-7 (n = 2) 7-10 (n = 6) (n = 9) (n = 11) (n = 8) ANOVA (n = 8) Probability E z (pg/ml) P (ng/ml) CA-125 (U/mL) K562-PB:j: K562-PV 49.5 ± 53t 1.5 ± 1.4 9,788 ± 8, ± ± ± ± 0.6 3,692 ± 2, ± ± ± ± 0.3 2,518 ± 1, ± ± 9.3 1,128 ± 1, ± 135 2,390 ± 2, ± ± 6.5 1,329 ± 1, ± 112 1,804 ± 1, ± ± ± ± ,185 ± 2, ± ± * Cycle day was determined according to the 1st day of their LMP during the follicular phase and by the BBT or by endometrial biopsy dating during the luteal phase. t Values are means ± SD. :j: K562-assay with PBMC, values are percent means ± SD of SIR. K562-assay with PFMC, values are percent means ± SD of SIR. Oosterlynck et al. NK activity in PF 293

5 mechanism. Viable endometrial cells are found in >50% of PFs of women whether endometriosis is present or not (17). More recently, Kruitwagen et al. (18) quantified the amount of refluxed endometrial cells by measuring the number of endometrial epithelial cell colonies in cultures of uterine-tubal flushings. In 92% of the cultures these colonies were identified, although no differences were found in the number of developing colonies between women with and without endometriosis. We reported a decreased cytotoxicity of autologous peripheral lymphocytes to endometrial cells in women with endometriosis (10). These results were recently confirmed by Vigano et al. (19). We delineated the NK cell as the responsible effector cell for the in vitro killing of endometrial cells because after depletion of the NK cells, using the anti-leu-lib monoclonal antibody, the cytotoxicity toward the endometrial cells decreased almost completely (10). In addition, we found a decreased peripheral NK activity in women with endometriosis, and this correlated with the decreased cytotoxicity to autologous endometrial cells. Vigano et al. (19) found also a decreased cytotoxicity to K562 cells in women with endometriosis, although the differences were not statistically significant. That the NK cell may be responsible for the cytotoxicity to ectopic endometrial implants is plausible. Natural killer cells are an integral component of the host defense system, and it is known that natural cytotoxicity is playing a central role in surveillance against tumor development. It is tempting to hypothesize that the NK cells of the PF are the first line defense mechanism against the implantation of endometrial cells in the peritoneal cavity. Hill et al. (11) already described with immunohistologic techniques the presence of NK cells in the PF. Our study was conducted to investigate the functional activity of these NK cells in the peritoneal cavity. In PF, an important NK activity was found, and this NK-mediated cytotoxicity was significantly decreased in women with endometriosis. The more severe the endometriosis, the lower the NK activity in the PF; moreover, this defect was 2.4 times higher than the defect found in PB. Furthermore, in women with endometriosis the PF NK activity is significantly decreased in the follicular phase of the cycle compared with the postovulatory phase. This could be an important pathophysiological finding. First, more endometrial cells are found during the follicular phase whether endometriosis is present or not (18). Second, implantation of those endometrial cells, reaching the peritoneal cavity by retrograde menstruation, will likely occur during the follicular 294 Oosteriynck et al. NK activity in PF phase of the cycle because viability of those cells will decrease afterward. Recapitulating the present findings, the local NK activity is decreased in women with endometriosis and this first-line defense mechanism is even more deficient at the moment when endometrial cells reach the peritoneal cavity. Confirming other reports (2-7), we found an increased number and concentration of PFMC in women with endometriosis, although the differences were not significant. To correct for this differences, the total NK activity and the NK activity per ml of PF were measured. Both the total NK activity and the NK activity per ml were still decreased significantly in relation to the severity of the disease. Furthermore, the decreased NK activity in the PF of women with endometriosis could not be attributed to a difference in the ratio of macrophages versus lymphocytes as shown on the smears of the PFMC in the final suspension. Moreover, using nonadherent cells, containing relatively more lymphocytes, no obvious changes in NK activity of the PFMC could be found. This demonstrated that the macrophages do not inhibit the lymphocytes in their NK cytotoxicity. On the other hand, these depletion experiments were performed on women without endometriosis, and it has been suggested that the peritoneal macrophages of women with endometriosis have an increased activation (4-6). Therefore, further experiments are needed to investigate the possible inhibitory effects of the macrophages of women with endometriosis on the lymphocyte cytotoxicity. We found a significant correlation between the color of the PF and the severity of endometriosis, in accordance with the findings of Halme et al. (20). The presence of RBCs in the PF is to be seen as a physiological condition. This can be because of retrograde menstruation, especially because we found more frequently red-colored fluids in the proliferative phase of the cycle. Kruitwagen et al. (18) found also more endometrial epithelial cell colonies when uterine tubal flushing was performed during the early follicular phase. We found a significant correlation between the cycle day and the color of the fluid but not between the cycle day and the severity of endometriosis. For this reason, to be sure we had a representative population, we excluded from our study only the very bloody fluids (n = 5) because of the risk of contamination with PBMC. But even when those women were included, of whom three had endometriosis and two did not, the main findings were confirmed. The next question to answer is why the NK activity is decreased in the PF of women with endometriosis. In our first study (10), we demonstrated Fertility and Sterility

6 with flow cytometry that the NK deficiency of PBMC was a functional defect rather than a quantitative defect because the number and percentages of the lymphocyte subsets were not different between women with endometriosis and women without endometriosis. We have some evidence that the decreased NK activity of PBMC is a primary defect, present before the development of endometriosis, because repeating the immunological assays after CO 2 laser excision of the endometriotic lesions no increase of the cytotoxicity assays was found (Oosterlynck et ai., unpublished observations, 1992). The decrease in NK activity in the PF of women with endometriosis is likely to be because of a functional deficiency because Hill et ai. (11) found an increase in the concentration of NK cells in the PF of women with endometriosis. Further experiments will be needed to investigate the possible in vitro immunosuppressive effect of PF on NK activity. In the present study, we found a lower NK activity of the PFMC during the follicular phase compared with the postovulatory phase in women with endometriosis. If this lower activity during the follicular phase is because of alterations in hormone concentrations or because of a possible inhibitory effect of the endometrial cells is still to be elucidated. The latter could be true because using peripheral heterologous control lymphocytes in a previous study (10), the endometrial cells of women with endometriosis showed a resistance to NK-mediated cytotoxicity. In conclusion, the NK activity of the PFMC in women with endometriosis was 2.4 times lower than in women without endometriosis. This can be the reason for an impaired clearance of the endometrial cells that reach the peritoneal cavity in those women. Acknowledgments. The authors thank Mrs. Marie Josee Vanderheyden for her excellent technical assistance, Mr. Jozef Goebels for culturing the tumor cell line, and Mrs. Diane W olput for typing this manuscript. REFERENCES 1. van Furth R, Raeburn JA, van Zwet TL. Characteristics of human mononuclear phagocytes. Blood 1979;54: Haney AF, Muscato JJ, Weinberg JB. Peritoneal fluid cell populations in infertility patients. Fertil Steril 1981;35: Halme J, Becker S, Hammond MG, Raj S. Pelvic macrophages in normal and infertile women: the role of patent tubes. Am J Obstet GynecoI1982;142: Muscato JJ, Haney AF, Weinberg JB. Sperm phagocytosis by human peritoneal macrophages: a possible cause of infertility in endometriosis. Am J Obstet Gynecol 1982;144: Badawy SZ, Cuenca V, Marshall L, Munchback R, Rinas AC, Coble DA. Cellular components in peritoneal fluid in infertile patients with and without endometriosis. Fertil Steril 1984;42: Dunselman GA, Hendrix MG, Bouckaert PX, Evers JL. 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